The aryl hydrocarbon receptor (AHR) mediates the toxicological effects of structurally diverse ligands, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). Properties of the AHR, including its ligand binding affinity, can underlie wide variations in the relative potency of different agonists and the sensitivity of different animal groups. Frogs are extremely insensitive to TCDD toxicity, and AHRs from Xenopus laevis (African clawed frog) bind TCDD with >20-fold lower affinity than the AHR from a highly sensitive mouse strain. In addition to its role in xenobiotic toxicity, the AHR may also mediate the effects of natural ligands. One candidate endogenous ligand is 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct. In the previous grant period, we showed FICZ is a potent AHR agonist in a frog cell line--at least 30-fold more potent than TCDD for cytochrome P4501A (CYP1A) induction. This suggests that FICZ binds frog AHRs with higher affinity than TCDD. FICZ potency declined with time in culture, suggesting that unlike TCDD, it is metabolized by enzymes it induces. These two properties are consistent with the hypothesis that although frog AHRs have lost the ability to bind TCDD with high affinity, they nonetheless exhibit high responsiveness to a putative physiological ligand. In this AREA grant renewal application, we propose an integrated set of experiments that logically follow our previous characterizations of frog AHR signaling. This project will probe the structure and effects of frog AHRs in conjunction with both TCDD and FICZ, undertaking three specific aims: (1) Guided by a recently developed homology model of the X. laevis AHR ligand binding domains, we will use site-directed mutagenesis to make the frog AHRs more "mouse-like," determining which differences confer low TCDD affinity. We will also test the hypothesis that determinants of FICZ binding differ from those of TCDD. These studies will identify important structural features of AHR's ligand binding pocket with both natural and xenobiotic ligands. (2) Using RNA-seq, we will test the hypothesis that FICZ and TCDD elicit expression changes in distinct sets of target genes. This highly sensitive and quantitative approach will improve on preliminary Affymetrix microarray studies by enabling the identification of the many unknown transcripts on commercial arrays, filling an enormous annotation gap in Xenopus genomics. (3) We will determine the catalytic specificity of CYP1A6 and CYP1A7, evolutionarily unique CYP1A paralogs. These studies will test the hypothesis that key amino acid differences in the catalytic domains of each enzyme confer substrate preferences, including specificity for FICZ metabolism. Overall, our comparative approach treats the unique features of the frog AHR signaling pathway as "mutant phenotypes" to glean important general information about the toxicological and biological functions of this system in humans and other vertebrates. Understanding the differences between frog and human AHRs will also aid risk assessment by refining interpretation of toxicological data derived from FETAX and similar developmental toxicity tests employing frog embryos. PUBLIC HEALTH RELEVANCE: This project will study the aryl hydrocarbon receptor (AHR), a protein that mediates the toxic effects of environmental contaminants such as dioxin. This protein also has important roles in physiology and development which we seek to better understand. We will compare the structure, function, and downstream effects of the AHR from frogs, which are insensitive to dioxin toxicity, with AHRs from more sensitive animals like mice and humans. This comparative research will help scientists identify the most important features of this system that are shared between all vertebrates. It will also aid in the understanding of important differences between frogs and humans as they interpret toxicology studies using frog embryos as a model system for measuring the effects of chemicals and environmental samples.